The principal function identified for the CF gene product, CFTR, is that of a cAMP-dependent C1-channel. The most serious medical problem faced by CF patients is debilitating and eventually fatal lung disease. Our broad, long term is to determine if mutations in the CF gene lead to lung disease by decreasing airway epithelial cAMP-dependent C1- conductance. Much has been learned about the C1-conductance mediated by CFTR, and mutations in the CF gene appear to affect CFTR function in one of two ways. First, the most common mutation of the CF gene, deletion of a phenylalanine at position 508, may cause misfolding of CFTR during protein synthesis so that it never reaches the plasma membrane. Second, mutations in the CF gene may abolish or diminish the functional capacity of CFTR molecules. This appears to be the case for certain mutations, which, when paired with the deletion of phenylalanine at 508, cause a milder clinical course of CF disease. To date, the available techniques are not sufficient to sort out the relative importance of these mechanisms. Part of the difficulty is the lack of specificity and sensitivity of reagents to document CFTR distribution within the cell, especially the fraction of normal and mutant CFTRs in the plasma membrane. Project IA of the SCOR will develop new antibodies against human CFTR that will improve our ability to determine if mutations in the CF gene prevent CFTR from reaching the plasma membrane. Another difficulty is that the molecular details of how CFTR serves as a C1- channel are not completely known, and thus prevent us from understanding the impact of mutations that may affect CFTR function. In Specific Aim 1 of this application we will use site directed mutagenesis and heterologous expression of recombinant CFTR mutants to address the roles of putative transmembrane spanning segments, nucleotide binding folds and CFTR cystienes in C1- conductance. In specific Aim 2 we will address how much CFTR needs to be present in the plasma membrane to provide a normal level of cAMP-dependent conductance. We will use cells derived from mice or patients who have mutations that disrupt translation from one or both alleles of the CF gene, and therefore, compared to normals, should transcribe 50% and 0% full length CFTR. First, we will use mice with one allele of the CF gene disrupted and one intact as a model for CF heterozygotes. We will compare how much CFTR is transcribed, processes to the plasma membrane, and expressed as cAMP-dependent C1- conductance. Finally, we will determine the relationship between mild CFTR mutants and cAMP-dependent C1- conductance in polarized airway epithelia of transgenic animals engineered in Project IA.